ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide-inactivating and nucleotide-phosphorylating ecto-enzymes. We suggest that extracellular ATP homeostasis in pancreatic ducts may be important in pancreas physiology and potentially in pancreas pathophysiology.

MicroRNA-155 modulates P2R signaling and Th2 priming of dendritic cells during allergic airway inflammation in mice.

BACKGROUND: Dendritic cells (DCs) are the professional antigen-presenting cells (APCs) in the lung. They are known to be key players in the induction and maintenance of allergic asthma by cross-linking innate and adaptive immune responses. MicroRNAs (miRNAs) are known to influence cell fate and function by translational suppression or induction of messenger RNA (mRNA) degradation. miR-155 has been shown to be a crucial regulator of the immune system. However, its function in the pathogenesis of allergic airway inflammation (AAI) is not completely elucidated yet. METHODS: Wild type (WT) and miR-155-deficient (miR-155(-/-) ) mice were used in ovalbumin (OVA) and house dust mite (HDM) models of AAI. Adoptive transfer of sensitized DCs to the lungs, migration, and T-cell priming assays were used to investigate the functional relevance of miR-155 in DCs. RESULTS: miR-155(-/-) mice showed reduced eosinophilic airway inflammation compared to WT mice in both models of AAI. Furthermore, miR-155(-/-) DCs showed limited Th2 priming capacity and failed to induce airway inflammation in allergen-exposed WT mice. miR-155 deficiency on DCs was also associated with impaired purinergic receptor signaling, as miR-155(-/-) DCs showed reduced chemotaxis and IL-1beta secretion upon stimulation with ATP, probably due to direct targeting of ectonucleoside triphosphate diphosphohydrolases (ENTPD) by miR-155. CONCLUSIONS: miR-155 deficiency alleviates AAI by diminishing Th2 priming capacity and ATP-/P2R-induced activation of DCs in mice, suggesting this miRNA as a potential therapeutic target of AAI.

A cell surface amine oxidase directly controls lymphocyte migration.

Lymphocytes leave the blood using a sequential adhesion cascade. Vascular adhesion molecule-1 (VAP-1) is a surface-expressed endothelial glycoprotein, which belongs to a distinct subgroup of monoamine oxidases. We show here that catalytic activity of VAP-1 on primary endothelial cells directly regulates lymphocyte rolling under defined laminar shear. VAP-1 seems to bind to a primary amino group presented on the lymphocyte surface and oxidatively deaminate it in a reaction, which results in the formation of a transient covalent bond between the two cell types. Instead, soluble reaction products (aldehydes and hydrogen peroxide) are not needed for the VAP-1-dependent rolling. Enzymatic regulation of lymphocyte adhesion to endothelium provides a previously unrecognized rapid way of controlling the extravasation process.

Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions.

Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP --> ADP --> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.

Circulating soluble vascular adhesion protein 1 accounts for the increased serum monoamine oxidase activity in chronic liver disease.

BACKGROUND & AIMS: Vascular adhesion protein 1 (VAP-1) is an endothelial glycoprotein that supports adhesion of lymphocytes to hepatic endothelium and has sequence homology with semicarbazide-sensitive amine oxidases (SSAOs). We investigated whether soluble VAP-1 (sVAP-1) displays SSAO activity and thereby accounts for increased monoamine oxidase activity in the serum of patients with liver diseases. METHODS: sVAP-1 concentration and SSAO activity were measured in peripheral, hepatic, and portal blood and in bile from patients with liver disease and in peripheral blood of control subjects, using enzyme-linked immunosorbent assay and enzymatic assays. RESULTS: sVAP-1 concentration (mean [+/-SE], 143. 67 [34.97-92.67] ng/mL) and SSAO activity (18.8 [12.0-24.6] nmol. mL(-1). h(-1)) were significantly increased in chronic liver diseases compared with healthy controls (87.1 [53.5-127] ng/mL [P<0.001] and 10.7 [6.5-12.7] nmol. mL(-1) x h(-1) [P<0.05]) but not in massive necrosis caused by paracetamol poisoning (109 [80.3-140] ng/mL and 8.9 [5.7-12.3] nmol. mL(-1) x h(-1)). sVAP-1 correlated with serum transaminase and bilirubin but not with creatinine. In 5 paired samples, sVAP-1 concentration was higher in hepatic (median, 113 [range, 53-122]) than in portal vein (102 [42-109]; 2P<0.05), and was not detected in bile. There was a highly significant correlation between serum sVAP-1 and SSAO activity in normal subjects, patients with acute liver failure, and those with chronic liver disease (r = 0.895; P<0.001). When serum was depleted of sVAP-1 by immunoaffinity chromatography, SSAO activity was eliminated. CONCLUSIONS: sVAP-1 levels are increased in chronic liver disease, and sVAP-1 is likely derived from the liver. Serum sVAP-1 displays SSAO activity and accounts for most of the monoamine oxidase activity in human serum.

Inhibitory effects of some purinergic agents on ecto-ATPase activity and pattern of stepwise ATP hydrolysis in rat liver plasma membranes.

Inhibitory effects of various purinergic compounds on the Mg(2+)-dependent enzymatic hydrolysis of [(3)H]ATP in rat liver plasma membranes were evaluated. Rat liver enzyme ecto-ATPase has a broad nucleotide-hydrolyzing activity, displays Michaelis-Menten kinetics with K(m) for ATP of 368+/-56 microM and is not sensitive to classical inhibitors of the ion-exchange and intracellular ATPases. P2-antagonists and diadenosine tetraphosphate (Ap(4)A) progressively and non-competitively inhibited ecto-ATPase activity with the following rank order of inhibitory potency: suramin (pIC(50), 4.570)>Reactive blue 2 (4.297)&z.Gt;Ap(4)A (3. 268)>pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (2. 930). Slowly hydrolyzable P2 agonists ATPgammaS, ADPbetaS, alpha, beta-methylene ATP and beta,gamma-methylene ATP as well as the diadenosine polyphosphates Ap(3)A and Ap(5)A did not exert any inhibitory effects on the enzyme activity at concentration ranges of 10(-4)-10(-3) M. Thin-layer chromatography analysis of the formation of [(3)H]ATP metabolites indicated the presence of other enzyme activities on liver surface (ecto-ADPase and 5'-nucleotidase), participating in concert with ecto-ATPase in the nucleotide hydrolysis through the stepwise reactions ATP-->ADP-->AMP-->adenosine. A similar pattern of sequential [(3)H]ATP dephosphorylation still occurs in the presence of ecto-ATPase inhibitors suramin, Ap(4)A and PPADS, but the appearance of the ultimate reaction product, adenosine, was significantly delayed. In contrast, hydrolysis of [(3)H]ATP in the presence of Reactive blue 2 only followed the pattern ATP-->ADP, with formation of the subsequent metabolites AMP and adenosine being virtually eliminated. These data suggest that although nucleotide-binding sites of ecto-ATPase are distinct from those of P2 receptors, some purinergic agonists and antagonists can potentiate cellular responses to extracellular ATP through non-specific inhibition of the ensuing pathways of purine catabolism.

Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5'-nucleotidase along with endogenous ATP from vascular endothelial cells.

Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5'-nucleotidases). The activity of soluble ATPase was progressively increased from 1.62+/-0.27 to 12.7+/-1.0 pmoles ml(-1) h(-1) after 60 min of stimulation by shear stress. The rate of [(3)H]-ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid, and remained insensitive to the classic inhibitors of ion-pumping and intracellular ATPases. Shear stress also increased the activity of 5'-nucleotidase in the medium from 2.0+/-0.5 to 27.2+/-2.8 pmoles ml(-1) h(-1). When shear stress was applied after removal of ecto-5'-nucleotidase by phosphatidylinositol-specific phospholipase C, the release of 5'-nucleotidase was drastically reduced. These results show that soluble ATPase and 5'-nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.

Human vascular adhesion protein-1 in smooth muscle cells.

Human vascular adhesion protein-1 (VAP-1) is a dual-function molecule with adhesive and enzymatic properties. In addition to synthesis in endothelial cells, where it mediates lymphocyte binding, VAP-1 is expressed in smooth muscle cells. Here we studied the expression, biochemical structure, and function of VAP-1 in muscle cells and compared it to those in endothelial cells. VAP-1 is expressed on the plasma membrane of all types of smooth muscle cells, but it is completely absent from cardiac and skeletal muscle cells. In tumors, VAP-1 is retained on all leiomyoma cells, whereas it is lost in half of leiomyosarcoma samples. In smooth muscle VAP-1 predominantly exists as a approximately 165-kd homodimeric glycoprotein, but a trimeric (approximately 250 kd) form of VAP-1 is also found. It contains N-linked oligosaccharide side chains and abundant sialic acid decorations. In comparison, in endothelial cells dimeric VAP-1 is larger, no trimeric forms are found, and VAP-1 does not have N-glycanase-sensitive oligosaccharides. Unlike endothelial VAP-1, VAP-1 localized on smooth muscle cells does not support binding of lymphocytes. Instead, it deaminates exogenous and endogenous primary amines. In conclusion, VAP-1 in smooth muscle cells is structurally and functionally distinct from VAP-1 present on endothelial cells.

Steady-state binding of adenine nucleotides ATP, ADP and AMP to rat liver and adipose plasma membranes.

Binding of native adenine nucleotides to rat liver and adipose plasma membranes was studied under steady-state conditions using EDTA/Na for inhibition of ecto-nucleotidase activity. [3H]-labelled ATP, ADP and AMP are able to interact with specific binding sites with respective Kd values of 88 +/- 9, 278 +/- 29 and 495 +/- 40 nmol/l for liver membranes; and of 64 +/- 7, 231 +/- 36 and 2050 +/- 290 nmol/l for adipose membranes. The nucleotide-binding capacity (Bmax) varied from 15 to 18 pmol/mg protein in the case of [3H]ATP and [3H]ADP-binding studies and from 22 to 26 pmol/mg protein for [3H]AMP-binding sites. Both 2-MeSATP and ADP inhibited [3H]ATP-binding to membranes with respective IC50 values of 60 +/- 7 and 285 +/- 30 nM. Other purinergic agents suramin, Reactive blue 2, alpha,beta-MeATP and beta,gamma-MeATP were less potent competitors of [3H]ATP binding, whereas AMP, adenosine, GTP, UTP, and CTP did not cause any displacement effect at concentrations of 10(-6)-10(-5) M. It is suggested that the described ATP/ADP-binding sites are linked to G protein-coupled P2Y receptors, whereas AMP-binding sites may represent a substrate-binding component of the membrane ecto-5'-nucleotidase.

Steady-state binding of [3H]ATP to rat liver plasma membranes and competition by various purinergic agonists and antagonists.

Steady-state analysis of nucleotide-binding sites on rat liver plasma membranes was carried out using 3H-labelled ATP as radioligand under complete inhibition of ecto-ATPase activity by excess EDTA. Binding of [3H]ATP to the membranes is saturable, reversible and apparently involves one population of specific binding sites with Kd of about 90 nM and binding capacity (Bmax) of 15 pmol/mg protein. A broad spectrum of purinergic agonists and antagonists was examined as potential inhibitors of the measured binding. The displacement studies showed the following rank order of inhibitory potency for [3H]ATP-binding sites (pIC50 values in parentheses): ATPgammaS (7.49)>2-MeSATP (7.18)>ATP (6.91)>ADPbetaS (6.64)>/=ADP (6.56)>>RB2 (6.14)>>suramin (5.40)>>Ap4A (4. 57)>alpha,beta-MeATP (4.19)>/=beta,gamma-MeATP (3.97). AMP, adenosine, Ap5A, PPADS, beta-glycerophosphate as well as non-adenine nucleoside triphosphates GTP, UTP and CTP did not exert any effect on the measured binding at concentration ranges of 10-6-10-4 M. In order to ascertain whether ATP and its analogues are capable of interacting with the same binding domain, 2-MeSATP and ADP were treated as alternative ligands that could compete with unlabelled ATP for its binding sites. A 2-fold increase of Kd value for ATP-receptor interaction was observed in the presence of 2-MeSATP (60 nM) or ADP (250 nM) without any modulation of Bmax value, confirming that inhibitory effects of these compounds are competitive in nature. These studies demonstrate that ATP and its analogues are able to interact with a single binding domain on liver plasma membranes, which may be identified as ligand-binding component of P2 purinoceptors of the P2Y1 subtype.

Kinetic analysis of enzymatic hydrolysis of ATP in human and rat blood serum.

Kinetic analysis of enzymatic hydrolysis of 14C- or 3H-labelled ATP was performed in the blood of healthy men donors and male Wistar rats. Nonspecific phosphatases were paralleled in blood serum by specific enzyme ATPase which is capable of dephosphorylating exogenous ATP in cooperation with other serum nucleotidases ultimately to adenosine. APparent Michaelis-Menten constant (Km) and maximal velocity (Vmax) values for rat serum ATPases are 68 +/- 7 microM and 7.0 +/- 0.3 nmoles ATP/mg protein per h, respectively. ATPase from human blood serum was characterized by lower respective Km and Vmax values: 39 +/- 5 microM and 2.5 +/- 0.2 nmoles ATP/mg protein per h. Activity of serum ATPase was decreased in the presence of membrane ecto-ATPase inhibitors PPADS and RB2, whereas the Na,K-ATPase inhibitor ouabain did not exert any inhibitory action on the measured enzyme activity.

Effects of triton X-100 and concanavalin A on the properties of 5'-nucleotidase in rat liver and adipose plasma membranes: a role of membrane structure in the regulation of enzyme activity.

The kinetic and thermodynamic properties of 5'-nucleotidase (EC 3.1,3.5) were investigated in rat liver and adipose plasma membranes (PM) after their structural modification with nonionic detergent Triton X-100 (TX-100) or lectin concanavalin A (Con-A). The apparent K(m) value of 5'-nucleotidase decreased after PM treatment with subsolubilizing TX-100 concentrations (0.005-0.015%) and then tended to grow at higher TX-100 concentrations (0.03-0.05%). Treatment of PM with Con-A resulted in the noncompetitive inhibition of 5'-nucleotidase in a dose-dependent manner. The Arrhenius plot of the 5'-nucleotidase activity in the control PM exhibited a single well defined break at about 28-31 degrees C with a lower activation energy at the upper slope of the graph. The shape of the Arrhenius graphs and the thermodynamic parameters of 5'-nucleotidase were specifically modified upon the PM treatment with increasing concentrations of TX-100 or Con-A. It was suggested that the functional activity of 5'-nucleotidase and its conformation within the membrane matrix may be directly regulated by the structural states of the enzyme specific microenvironment and of the membrane lipid bilayer. Moreover, the existence of the spatial and/or compositional optima for the relationship between the enzyme molecule and its lipid surrounding may be reasonably supposed.

Effect of increasing concentrations of nonionic detergent Triton X-100 on solubilization and structure of rat liver and adipose plasma membranes.

The extent of solubilization and the structure of rat liver and adipose plasma membranes after treatment with nonionic detergent Triton X-100 were studied. The concentration of Triton X-100 varied from 0.005 to 0.050% (0.26-2.6 mg/mg membrane protein). The excimerization of the pyrene fluorescent probe and the relative vibronic band intensities in the pyrene monomer fluorescence spectrum were measured to evaluate the membrane lipid bilayer fluidity and polarity. The data on the aqueous pyrene fluorescence in the presence of Triton X-100 showed that formation of micellar aggregates occurred at detergent concentrations of over 0.015%. This value is a crucial factor in the detergent action on the membrane structure: both intensive extraction of plasma membrane proteins and fluidization of the lipid bilayer were observed only at Triton X-100 concentration over 0.015%. However, Triton X-100 did not exert any action on the polarity of the membrane hydrophobic regions.

Characterization of [3H]AMP binding to rat adipose plasma membranes and its substrate specificity.

We evaluated the binding of [3H]AMP to rat adipose plasma membranes and studied the substrate specificity of the process. The [3H]AMP binding was investigated by high-speed filtration under conditions of virtually complete inhibition of the 5'-nucleotidase activity by EDTA/Na. The Scatchard plot revealed the existence of a single class of AMP-binding sites on the membrane surface with Kd of 2.14 +/- 0.210 microM and the binding capacity (Bmax) of 26.0 +/- 0.68 pmol per mg protein. Addition of ATP (12.5 microM) or ADP (3.5 microM) to the incubation medium resulted in a two-fold increase of Kd, whereas in the presence of adenosine (400 microM) or its pharmacological antagonist theophylline (200 microM), the number of AMP-binding sites decreased. Therefore, ATP and ADP but not adenosine compete with AMP for the same nucleotide-binding site. It is suggested that the observed [3H]AMP binding may be primarily caused by the interaction of AMP with a specific substrate-binding active centre of the membrane ectoenzyme 5'-nucleotidase.

Alteration of 5'-nucleotidase properties in rat adipose and liver plasma membranes after 1 Gy whole-body gamma-irradiation.

5'-Nucleotidase activities in rat adipose and liver plasma membranes (PM) were examined at various times after 1 Gy whole-body gamma-irradiation. Changes in the enzyme kinetic parameters were recorded 6 and 14 weeks after exposure: Km decreased while Vmax increased. The enzyme in control PM showed temperature-dependent Arrhenius plots with a lower activation energy at temperatures above a break point at approximately 28 degrees C, whereas 5'-nucleotidase in irradiated PM showed a constant activation energy between 10 and 40 degrees C which was reasonably close to the value observed controls at temperatures below the break point. Furthermore, the interaction of [3H]-AMP with the 5'-nucleotidase catalytic site was studied on adipose PM using a rapid filtration technique. gamma-Irradiation was associated with an increased number of specific AMP-binding sites and decreased ligand affinity. Comparison of the presented data with previous work (Yegutkin et al. 1993a,b) indicate significant functional and structural modification of rat adipose and liver PM following 1 Gy gamma-irradiation and reveals interrelation of these changes.

Evaluation of age-related changes of physicochemical properties and functional activity of rat adipose plasma membranes and their possible relationship.

Studies were carried out to evaluate structural state of adipose plasma membranes (PM) from mature adult and aged Wistar rats and its possible relation to functional changes in aging. PM lipids were probed by fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene. The data on DPH anisotropy, pyrene excimerization and induction resonance energy transfer (IRET) from PM proteins to pyrene provided the evidence for age-dependent decrease in PM lipid phase fluidity inclusive of that of annular lipid. Interaction between PM lipids and integral proteins also changed in aging. The observed structural changes were due to age-related lipid compositional modification. Evidence for this conclusion was provided by the following data: (i) DPH anisotropy was increased in aging both in PM and liposomes from the same PM lipids; (ii) both saturation/unsaturation fatty acid ratio and relative content of phosphatidylethanolamine (PE) increased in aged PM, whereas cholesterol/phospholipid and lipid/protein ratios were age-independent; (iii) PM polypeptide composition remained practically unchanged during aging. Age-related changes of the PM structure were accompanied by a twofold decrease of high affinity insulin binding sites and elevation of their affinity to insulin. It was suggested that age-related changes of physicochemical properties of PM lipid phase might affect protein molecular conformation and function through either changes in bulk lipid fluidity or through specific lipid-protein interactions.